Project Summary The goal of this project is to determine how renal tubular epithelial cells achieve robust cell-cell adhesion when faced with external forces. In the kidney, this occurs regularly as volume fluctuations distend the urinary collecting system to varying degrees. An extreme example occurs in autosomal-dominant polycystic kidney disease (ADPKD), the most common inherited renal disorder, where renal cysts can endure 1000-fold strain in diameter, but can rupture upon acute trauma, leading to other serious consequences. Approximately 85% of ADPKD cases are caused by mutations in the protein polycystin-1 (Pc-1), a putative atypical G-protein coupled receptor that is involved in intracellular signal transduction via sequestration of the G protein subunit G?12. Relatively little is known about how dysregulated G?12-mediated signaling in ADPKD leads to the physical compromise of cell-cell adhesion. This gap persists, in part, because even simple questions remain unanswered about how epithelial cells mechanically regulate cell-cell adhesions under strain. This proposal will address two such fundamental questions, using the case of ADPKD as a concrete example of how such regulation may be disrupted. To do so, I will make use of a semi-reconstituted system in which Madin- Darby Canine Kidney (MDCK) epithelial cells form junctions with supported lipid bilayers (SLBs) decorated with the cell adhesion molecule E-cadherin. This system enables both high resolution microscopy on live cells and precise application of externally applied forces using magnetic tweezers. Aim 1 will address the question of how cells ensure robust adhesion using the E-cadherin molecules that bind between cells. High resolution total internal reflectance fluorescence (TIRF) and reflectance interference contrast (RICM) imaging will be used to visualize the clustering of E-cadherin and the cell-SLB distance, respectively, as a function of applied force. Aim 2 will address the question of how cells transmit external loads through the collection of E-cadherin molecules. Fluorescent single-molecule tension sensors will be used to directly measure single-molecule force distributions as a function of externally applied load. Finally, Aim 3 will systematically perturb the Pc-1/G?12 signaling axis to determine how cadherin-mediated adhesion and force transmission may be dysregulated in ADPKD. The results of this work will determine how signaling downstream of Pc-1 may contribute to the dysregulation of cell-cell adhesion in ADPKD, and, more broadly, reveal the biophysical mechanisms that epithelial cells use to maintain robust cell-cell adhesion even in the face of sometimes extreme external forces. When combined with a research training plan emphasizing development in research communication and incorporating continued clinical experience, this work will prepare me to pursue further training towards a career as an independent physician-scientist studying disrupted tissue architecture in disease.